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24876
Sequestosome Signaling Antibody Sampler Kit

Sequestosome Signaling Antibody Sampler Kit #24876

Western Blotting Image 1

Western blot analysis of extracts from MEF wt and U-2 OS cells, untreated (-) or treated with MG-132 #2194 (10 μM, 10 hr; +), using NRF2 (D1Z9C) XP® Rabbit mAb #12721.

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Western Blotting Image 2

Western blot analysis of extracts from NIH/3T3, NIH/3T3-TrkA, and NIH/3T3-TrkB using TrkA (12G8) Rabbit mAb (upper) TrkB (80E3) Rabbit mAb Antibody #4603 (middle) and PLCγ1 (D9H10) XP® Rabbit mAb #5690 (lower).

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Western Blotting Image 3

Western blot analysis of extracts from NIH/3T3, NIH/3T3-TrkA, and NIH/3T3-TrkB using TrkA (12G8) Rabbit mAb (upper) TrkB (80E3) Rabbit mAb Antibody #4603 (middle) and PLCγ1 (D9H10) XP® Rabbit mAb #5690 (lower).

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Western Blotting Image 4

Western blot analysis of seven distinct recombinant polyubiquitin chains (300 ng each) using K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb #5621 (upper) and Ubiquitin Antibody #3933 (lower).

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Western Blotting Image 5

Western blot analysis of extracts from various cell lines using SQSTM1/p62 (D5E2) Rabbit mAb #8025.

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Western Blotting Image 6

Western blot analysis of extracts from various cell lines using TRAF6 (D21G3) Rabbit mAb #8028.

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Western Blotting Image 7

Western blot analysis of extracts from various cell lines using KEAP1 (D6B12) Rabbit mAb #8047.

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Western Blotting Image 8

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® SQSTM1/p62 siRNA I #6394 (+) or SignalSilence® SQSTM1/p62 siRNA II #6399 (+), using SQSTM1/p62 (D5E2) Rabbit mAb (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The SQSTM1/p62 (D5E2) Rabbit mAb confirms silencing of SQSTM1/p62 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

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Western Blotting Image 9

Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with a cDNA expression construct encoding full-length human TRAF6 (+), using TRAF6 (D21G3) Rabbit mAb.

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Western Blotting Image 10

Western blot analysis of extracts from HeLa cells, untreated or treated with the proteasome inhibitor MG132 (10 µM for 6 hours), using K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb and Ubiquitin Antibody #3933 (lower).

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Western Blotting Image 11

Western blot analysis of extracts from NIH/3T3, NIH/3T3-TrkA, and NIH/3T3-TrkB using TrkA (12G8) Rabbit mAb (upper) TrkB (80E3) Rabbit mAb Antibody #4603 (middle) and PLCγ1 (D9H10) XP® Rabbit mAb #5690 (lower).

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Western Blotting Image 12

Western blot analysis of extracts from MEF wt and U-2 OS cells, untreated (-) or treated with MG-132 #2194 (10 μM, 10 hr; +), using NRF2 (D1Z9C) XP® Rabbit mAb.

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Western Blotting Image 13

Western blot analysis of extracts from various cell lines using KEAP1 (D6B12) Rabbit mAb.

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Western Blotting Image 14

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

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Western Blotting Image 15

Western blot analysis of extracts from various cell lines using SQSTM1/p62 (D5E2) Rabbit mAb.

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Western Blotting Image 16

Western blot analysis of extracts from various cell lines using TRAF6 (D21G3) Rabbit mAb.

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Western Blotting Image 17

Western blot analysis of seven distinct recombinant polyubiquitin chains (300 ng each) using K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb (upper) and Ubiquitin Antibody #3933 (lower).

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IHC-P (paraffin) Image 18

Immunohistochemical analysis of paraffin-embedded NIH/3T3-TrkA (left), NIH/3T3-TrkB (middle) and NIH/3T3-TrkC (right) cell pellets using TrkA (12G8) Rabbit mAb.

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IP Image 19

Immunoprecipitation of NRF2 from MEF wt cell extracts treated with MG-132 #2194 (10 μM, 10 hr) using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or NRF2 (D1Z9C) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using NRF2 (D1Z9C) XP® Rabbit mAb (lane 3).

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Chromatin IP Image 20

Chromatin immunoprecipitations were performed with cross-linked chromatin from MEF NRF2 wild-type (left) and NRF2 knock-out (right) cells, both treated with DEM (50 μM, 3 hr), and NRF2 (D1Z9C) XP® Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using mouse MafG intron 1 primers, SimpleChIP® Mouse NQO1 Promoter Primers #12635, and SimpleChIP® Mouse RPL30 Intron 2 Primers #7015. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

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Western Blotting Image 21

Western blot analysis of extracts from OVCAR8 cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® KEAP1 siRNA I #5285 (+) or SignalSilence® KEAP1 siRNA II #5289 (+), using KEAP1 (D6B12) Rabbit mAb (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The KEAP1 (D6B12) Rabbit mAb confirms silencing of KEAP1 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

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Western Blotting Image 22

Western blot analysis of extracts from SK-MEL-2 cells, untreated (-) or starved overnight in Earle's Balanced Salt Solution (EBSS) (+), using SQSTM1/p62 (D5E2) Rabbit mAb.

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Western Blotting Image 23

Western blot analysis comparing the titration of recombinant monoubiquitin, K48-linked polyubiquitin and K63-linked polyubiquitin using K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb (upper) and Ubiquitin Antibody #3933 (lower).

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IHC-P (paraffin) Image 24

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using TrkA (12G8) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

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Flow Cytometry Image 25

Flow cytometric analysis of MEF wt cells, untreated (blue) and treated with MG-132 #2194 (green), using NRF2 (D1Z9C) XP® Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.

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IF-IC Image 26

Confocal immunofluorescent analysis of OVCAR8 cells using KEAP1 (D6B12) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Western Blotting Image 27

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a tagged human SQSTM1/p62 construct (+), using SQSTM1/p62 (D5E2) Rabbit mAb.

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Western Blotting Image 28

Western blot analysis of various cell lines using K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb.

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IHC-P (paraffin) Image 29

Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using TrkA (12G8) Rabbit mAb.

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Western Blotting Image 30

Western blot analysis of extracts from HeLa cells using K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb, untreated or following antibody pre-incubation with either K63 ubiquitinylated branched peptide to block the signal or a linear peptide surrounding K63 of ubiquitin that cannot block the signal.

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
SQSTM1/p62 (D5E2) Rabbit mAb 8025 20 µl
  • WB
  • IP
H Mk 62 Rabbit IgG
TRAF6 (D21G3) Rabbit mAb 8028 20 µl
  • WB
  • IP
H Mk 60 Rabbit IgG
K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb 5621 20 µl
  • WB
All Rabbit IgG
TrkA (12G8) Rabbit mAb 2510 20 µl
  • WB
  • IHC
H 140 Rabbit IgG
NRF2 (D1Z9C) XP® Rabbit mAb 12721 20 µl
  • WB
  • IP
  • F
  • ChIP
H M Mk 97-100 Rabbit IgG
KEAP1 (D6B12) Rabbit mAb 8047 20 µl
  • WB
  • IF
H M R 60-64 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The Sequestosome Signaling Antibody Sampler Kit contains reagents to investigate sequestosome signaling within the cell. The kit contains enough antibodies to perform two western blot experiments per primary antibody.

Each antibody in the Sequestosome Signaling Antibody Sampler Kit detects endogenous levels of its target protein. K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb detects polyubiquitin chains formed by Lys63 residue linkage. It does not react with monoubiquitin or polyubiquitin chains formed by linkage to a different lysine residue. TRAF6 (D21G3) Rabbit mAb is not predicted to cross-react with other TRAF family members. TrkA (12G8) Rabbit mAb does not cross-react with TrkB.

Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly162 of human SQSTM1/p62 protein, residues near the amino terminus of human TRAF6 protein, residues surrounding the Lys63 branch of the human diubiquitin chain, residues surrounding Arg220 of human TrkA, residues surrounding Ala275 of human NRF2 protein, and residues near the carboxy terminus of human KEAP1 protein.

Sequestosome 1 (SQSTM1, p62) is a ubiquitin binding protein involved in cell signaling, oxidative stress, and autophagy (1-4). It was first identified as a protein that binds to the SH2 domain of p56Lck (5) and independently found to interact with PKCζ (6,7). SQSTM1 was subsequently found to interact with ubiquitin, providing a scaffold for several signaling proteins and triggering degradation of proteins through the proteasome or lysosome (8). Interaction between SQSTM1 and TRAF6 leads to the K63-linked polyubiquitination of TRAF6 and subsequent activation of the NF-κB pathway (9). Protein aggregates formed by SQSTM1 can be degraded by the autophagosome (4,10,11). SQSTM1 binds autophagosomal membrane protein LC3/Atg8, bringing SQSTM1-containing protein aggregates to the autophagosome (12). Lysosomal degradation of autophagosomes leads to a decrease in SQSTM1 levels during autophagy; conversely, autophagy inhibitors stabilize SQSTM1 levels. SQSTM1 also interacts with KEAP1, which is a cytoplasmic inhibitor of NRF2, a key transcription factor involved in cellular responses to oxidative stress (3). Under basal conditions, KEAP1 binds and retains NRF2 in the cytoplasm where it can be targeted for ubiquitin-mediated degradation (13). Small amounts of constitutive nuclear NRF2 maintain cellular homeostasis through regulation of basal expression of antioxidant response genes. Following oxidative or electrophilic stress, KEAP1 releases NRF2, thereby allowing the activator to translocate to the nucleus and bind to ARE-containing genes (14). The coordinated action of NRF2 and other transcription factors mediates the response to oxidative stress (15). Thus, accumulation of SQSTM1 can lead to an increase in NRF2 activity (3). KEAP1 also targets the down regulation of NF-κB activity by targeting IKKβ degradation (16). TrkA is a member of Trk receptor tyrosine kinases and is activated by NGF, which stimulates TrkA polyubiquitination (17,18). TrkA regulates proliferation and is important for development and maturation of the nervous system (19). SQSTM1 interaction with TRAF6 controls synthesis of K63 polyubiquititination on TrkA (18, 20). TrkA polyubiquitination is essential for neurotrophin-dependent receptor internalization, cell differentiation, and signaling (18).

  1. Geetha, T. et al. (2005) Mol Cell 20, 301-12.
  2. Kirkin, V. et al. (2009) Mol Cell 34, 259-69.
  3. Cullinan, S.B. et al. (2004) Mol Cell Biol 24, 8477-86.
  4. Seibenhener, M.L. et al. (2007) FEBS Lett 581, 175-9.
  5. Nguyen, T. et al. (2005) J Biol Chem 280, 32485-92.
  6. Segal, R.A. and Greenberg, M.E. (1996) Annu Rev Neurosci 19, 463-89.
  7. Komatsu, M. et al. (2010) Nat Cell Biol 12, 213-23.
  8. Jaiswal, A.K. (2004) Free Radic Biol Med 36, 1199-207.
  9. Bjørkøy, G. et al. (2006) Autophagy 2, 138-9.
  10. Joung, I. et al. (1996) Proc Natl Acad Sci USA 93, 5991-5.
  11. Sanchez, P. et al. (1998) Mol Cell Biol 18, 3069-80.
  12. Puls, A. et al. (1997) Proc Natl Acad Sci USA 94, 6191-6.
  13. Vadlamudi, R.K. et al. (1996) J Biol Chem 271, 20235-7.
  14. Wooten, M.W. et al. (2005) J Biol Chem 280, 35625-9.
  15. Wooten, M.W. et al. (2005) J Biol Chem 280, 35625-9.
  16. Bjørkøy, G. et al. (2005) J Cell Biol 171, 603-14.
  17. Komatsu, M. et al. (2007) Cell 131, 1149-63.
  18. Lee, D.F. et al. (2009) Mol Cell 36, 131-40.
  19. Pankiv, S. et al. (2007) J Biol Chem 282, 24131-45.
  20. Huang, E.J. and Reichardt, L.F. (2003) Annu Rev Biochem 72, 609-42.
Entrez-Gene Id
9817 , 4780 , 8878 , 7189 , 4914
Swiss-Prot Acc.
Q14145 , Q16236 , Q13501 , Q9Y4K3 , P04629
For Research Use Only. Not For Use In Diagnostic Procedures.

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